|
| Status |
Public on Aug 02, 2014 |
| Title |
MA rep3 |
| Sample type |
SRA |
| |
|
| Source name |
LDM: longissimus dorsi muscle
|
| Organism |
Sus scrofa |
| Characteristics |
breed: Jinhua pig
gender: female
age: 7 years old
|
| Growth protocol |
pigs aged 7 years old (MA) and 0.5 year old (Young)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA of each collected tissue was extracted using DNeasy Blood & Tissue Kit (Qiagen)
Each MeDIP library was subjected to the paired-end sequencing using Illumina HiSeq 2000 and in 50 bp read length. Five µg original DNA was isolated using E.Z.N.A.® HP Tissue DNA Midi Kit (OMEGA) and was sonicated to ~ 100 – 500 bp fragments with a Bioruptor sonicator (Diagenode). Then libraries were constructed using the Illumina Paired-End protocol consisting of end repair, <A> base addition and adaptor ligation steps, and were performed using Illumina’s Paired-End DNA Sample Prep kit following the manufacturer’s instructions. Adaptor-ligated DNA was immunoprecipitated by monoclonal anti-methylcytidine antibody. The specificity of the enrichment were confirmed by qPCR using SYBR green mastermix (Applied Biosystems) and primers for positive and negative internal control DNA of non-human samples were supplied in the Magnetic Methylated DNA Immunoprecipitation Kit (Diagenode). Cycling of qPCR validation was 95℃ 5 min, followed by 40 cycles 95℃ 15 s and 60℃ 1 min. The enriched fragments with methylation and 10% input DNA were purified on ZYMO DNA Clean & Concentrator-5 columns following the manufacturer’s instructions. DNA was eluted in 30 µl buffer EB and its concentration was measured. Enriched fragments was amplified by adaptor-mediated PCR in a final reaction volume of 50 µl consisting of 23 µl purified DNA, 25 µl Phusion DNA polymerase mix (NEB) and 2µl PCR primers. Amplification consisted of 94℃ 30 s, 10 cycles of 94℃ 30s, 60℃ 30 s, 72℃ 30 s then prolong with 5 min at 72℃ and products could be hold at 4℃. Amplifications quality and quantity were evaluated by Agilent 2100 Analyzer DNA 1000 chips purified by 2% agarose gel and eluted in 15 µl buffer EB. Ultra-high-throughput 50 bp pair-end sequencing was carried out using the Illumina HiSeq 2000 according to manufacturer instructions. Raw sequencing data were processed by Illumina base-calling pipeline.
|
| |
|
| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
MeDIP library for the paired-end sequencing in 50 bp read length
|
| Data processing |
Raw sequencing data were processed by Illumina base-calling pipeline. After filtering the low quality reads, the MeDIP-seq data were aligned to the UCSC pig reference genome (Sscrofa9.2) using SOAP2 (Version 2.21). The paired-end mapped reads were defined as fragments (BED file) that were used in further analysis.
Genome_build: Sscrofa9.2
Supplementary_files_format_and_content: The processed data is BED file, which contains chromsome, fragment start site and fragment end site
|
| |
|
| Submission date |
Sep 09, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Mingzhou Li |
| E-mail(s) |
mingzhou.li@163.com
|
| Phone |
86-835-2886000
|
| Organization name |
Sichuan Agricultural University
|
| Department |
College of Animal Science and Technology
|
| Lab |
Institute of Animal Genetics and Breeding
|
| Street address |
No.46 Xinkang Road
|
| City |
Ya'an |
| State/province |
Sichuan |
| ZIP/Postal code |
625014 |
| Country |
China |
| |
|
| Platform ID |
GPL11429 |
| Series (1) |
| GSE50716 |
Genome-wide DNA methylation changes in skeletal muscle between young and middle-aged pigs [methylation] |
|
| Relations |
| BioSample |
SAMN02352623 |
| SRA |
SRX347645 |
